This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Our use of the mass spectrometry core facility will be to support our efforts in proteinase inhibitor discovery, characterization, and design. Our lab studies three major classes of proteinases as potential therapeutic targets: serine (membrane type serine protienase 1, kaposi's sarcoma herpes virus proteinase, and the granzymes), cysteine (cruzain, SARS major proteinase, falcipain), and aspartyl proteases (eqolysin, and HIV proteinase). Our work involves developing robust expression systems, enzymatic and biochemical characterization, determining peptide substrate specificity, synthesizing effective fluorogenic substrates, developing high-throughput screening assays, screening candidate inhibitor libraries, synthesizing novel inhibitors, and characterizing the mechanism of action of these inhibitors kinetically and structurally. The use of the mass spectrometry core facility is integral to our efforts. Mass spectrometry provides an orthogonal and definitive way of confirming that a given inhibitor forms a covalent adduct in the expected manner with the proteolytic enzyme under investigation.